The purpose of this study was to investigate the knowledge, attitudes, and behaviors of Greek undergraduate students on hand hygiene and mobile phone hygiene, in relation to their mobile phones’ microbial load. An anonymous self-reported questionnaire was distributed among 100 students and swabs were taken from their devices for microbiological cultures and for an on-site bioluminescence microbial load assessment. Hand washing was considered the most effective personal protective behavior by the majority (89%), while spatial restrictions (40%) and forgetfulness (31%) were the main reasons for neglecting hand washing. Most students washed their hands 6–10 times/day (32%) for approximately 11–20 s (35%). Although most devices were cleaned/disinfected within the last week (28%), many were contaminated with Staphylococcus saprophyticus (84%), Staphylococcus aureus (13%), and Escherichia coli (39%), while 75% of the samples exceeded the “fail” threshold limit of the luminometer’s measurements. Moreover, statistically significant differences (p < 0.05) were found between the devices’ microbial load and certain hand hygiene behaviors and preferences. The median cfu/cm2 of Escherichia coli was higher among first-year students of health sciences, while Staphylococcus aureus was more prevalent in females’ than in males’ mobile phones. It w therefore understood that undergraduate students’ knowledge, attitudes, and behaviors on hand hygiene and mobile phone hygiene are highly intertwined with the microbial load on their mobile phones.